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PEX11 promotes peroxisome
division independently of peroxisome metabolism
Xiaoling Li and Stephen J. Gould
Department of Biological Chemistry, The John Hopkins
University School of Medicine, Baltimore, MD 21205
Review
by Nicole Hesson
Summary and Critique
Peroxisomes
are membrane bound organelles that import all of their protein, and are
important in many different lipid metabolism, especially fatty acid β-oxidation. Studies have shown that PEX11 may be
involved in peroxisome metabolism or division.
PEX11 in humans has at least two forms, PEX11α and PEX11β. There are both known to be integral membrane
proteins, and the β form has been shown to increase the number of
peroxisomes in a cell. For this reason,
the authors choose to focus on PEX11β rather than both components.
In this paper, Li and Gould are trying to prove that
the PEX11 gene is responsible for “peroxisome division independent of
peroxisome metabolism.” First, they
performed a test to determine if PEX11 is sufficient for division
without growth stimulus. They grew the
cells in media lacking any growth promoters and overexpressed different
peroxisome genes. PEX11 was the
only gene that initiated peroxisome division.
The second hypothesis they tested was whether or not the effect of PEX11β
in the former experiment was dependent on normal peroxisome metabolism. They compared the growth of normal
fibroblast cells and cells lacking the PEX5 gene. PEX5-/- cells do not
perform normal metabolic functions because they do not transcribe mRNA. Therefore, the cells do not import proteins
into the lumen, nor do they β-oxidize fatty acids or synthesize ether
lipids. The over expression of PEX11β
had an increasing effect on the number of peroxisomes in both normal and
defective cells. They then tested if
the PEX11β gene was dependent on the media the cells were grown
on. On surroundings lacking
β-oxidation substrates, wild type cells contained twice as many
peroxisomes as the PEX11β-/- cells, revealing that “PEX11
proteins promote division regardless of the metabolic state of the peroxisomes”
(Li and Gould, 2002).
Figure 1: Cells in
panels A, C, and E have been microinjected with PEX11βmyc. These cells have been induced to over
express PEX11. Control cells in
panels B, D, and F have been microinjected with PMP34myc. PMP34 is a human homologue of another
yeast protein needed for β-oxidation.
Antibodies for the myc tag as well as PEX14 were used to detect indirect
immunoflourescence. PEX14 is a protein
normally found in peroxisome membranes.
Cells in A and B were incubated for 1.5 hours, C and D for 4.5 hours,
and E and F for 48 hours. At 1.5 hours,
peroxisome level is normal in both sets of cells. After 4.5 hours, there appear to be more peroxisomes in the PEX11β
cells. After 48 hours, it is clear that
PEX11β has had an effect on the experimental cells. The PMP34 cells showed no significant
increase in abundance of peroxisomes.
This figure is telling us that proliferation of peroxisomes does not
happen immediately. It takes at least
4.5 hours to show any significant difference.
This figure does a good job of proving to the reader what it is meant to
prove.
Figure 2: Normal cells
in B and C have been transfected with PMP34myc, while cells in D and E have
been transfected with PEX11βmyc.
Antibodies for the myc tag were used in panels B and D, while antibodies
for PEX14 were used in C and E. Other
PMP genes were also transfected, but they showed the same results as PMP34. Panel A simply quantifies the data shown in
B-E, that cells induced with PEX11β had a higher number of
peroxisomes. Figure 2 as a whole drives
home the fact that overexpression of any PMP is not responsible for an increase
in peroxisome, but it is in fact the over expression of PEX11β that
is causing the increase. While this
data does show that, it would have been nice to see either immunoflourescence
pictures or graph bars, or both, displaying the other PMP genes that had been
tested. This would have been more
convincing to prove that PEX11β is the gene responsible. Just showing one PMP example is not
enough. Several are needed.
Figure 3: Cells in B and C have been transfected with
PMP34myc, while cells in D and E have been transfected with PEX11βmyc. Antibodies for the myc tag were used in
panels B and D, while antibodies for PEX14 were used in C and E. The cells in this figure were not normal
cells. The peroxisomes in these cells
lacked the PEX5 gene. The PEX5
gene is an important gene for normal peroxisome metabolism. See above for details on PEX5
mutants. In PEX5 mutants
overexpressing the PEX11β gene, the number of peroxisomes was much
higher than untransfected cells (first bar in A), as well as those cells
transfected with PMP34myc (bar 2). PEX11β
cells are represented in bar 3. This
figure tells us that PEX11β can still promote peroxisome division
even if peroxisome metabolic actions are non functional – division and
metabolism are independent of one another.
Once again, it would have been good to see more PMP genes. It also would have been nice to see mutants
other than the PEX5 gene.
However, this figure is still fairly convincing.
Figure 4: Normal lab
strain yeast cells were made to express GFP/PTS1, which causes GFP to be
imported into the peroxisome lumen.
Panels A and B show the difference in GFP containing peroxisomes when
grown on different media. A was grown
on glucose, a sugar, and B shows cells grown on oleic acid, a fatty acid. Since peroxisomes are the sole site of fatty
acid oxidation in yeast cells, peroxisome number should increase on a fatty
acid, as shown in panel B. Cells in A
and B contain plasmids with theGAL1 promoter.
Panels C and D use a different strain of yeast. These cells have had the PEX11 gene
deleted, contain GFP/PTS1, and contain a high copy GAL1 promoter plasmid, but
the vectors do not induce any genes to be expressed. Cells in C were grown on a glucose media, and cells in D were
grown on a galactose media. There
appeared to be more peroxisomes in panel D, and this was due to the removal
from the glucose repression. Neither
glucose nor galactose, both sugars, showed as much peroxisome abundance as the
oleic acid in panel B. The first four
panels are control panels. Panel E
included cells with gal-induced expression of the PEX13 gene, and panel
F included cells with gal-induced expression of the YPR128C gene. PEX13 is involved in protein transport, and
YPR128C carries adenine to the membrane of the peroxisome. Both are PMPs. Neither E nor F displayed any more peroxisome quantity than panel
D. This data tells us that more than
PMP overexpression is necessary to increase peroxisome division. Panel G cells are similar to cells in panels
E and F, except the plasmid has been constructed to overexpress PEX11. These cells show peroxisome numbers close to
those seen in panel B. Since the cells
were not grown on a fatty acid media, this data suggests that PEX11 is
the cause of the peroxisome increase.
Panels H and I show cells with the PEX11 gene deleted, GFP/PTS1
added, a high copy GAL1 promoter plasmid, and the POX1 gene
deleted. This gene is the first step in
the β-oxidation pathway, and is essential for yeast cells to oxidize any
fatty acids. The GAL1 plasmid in panel
H induced expression of PEX13, while the plasmid in panel I induced
expression of PEX11. Panel H was
no different that panels E or F, while panel I exhibits peroxisome levels
similar to those seen in panels G and B.
Once more, this data shows us that PEX11 is sufficient to
increase the number of peroxisomes in a cell.
I did not really understand this figure at first, but now I think it is
very nice. They tested yeast strains
with one mutation, strains with more than one mutation, vectors with no genes,
vectors built to express different peroxisome genes, and strains grown on different
growth mediums. All data show us that PEX11β
is the gene that is causing the increase in peroxisome number, regardless of
number of mutations or growth media.
Figure 5: Panels B and
C contain wild type mouse fibroblasts, and panels D and E contain PEX11β-/-
cells. Both were grown on a normal
media as well as a serum free media, which contained no lipids or substrates
for the oxidation path. B and D were
used antibodies to PEX14, and C and E used antibodies to a matrix marker enzyme
catalase (? I assume this is a protein in the cell.). If PEX11β plays a role in oxidation, the number of
peroxisomes should be the same. If the
lipids and substrates are missing, the level of peroxisomes in wild type and
mutant should be low. If the gene is
responsible for division of peroxisomes, there ought to be a significant
difference in the levels of peroxisomes.
PEX11β will increase the number of peroxisomes regardless of
the media. This is exactly what we
see. The wild type cells show about
twice as many peroxisomes as the mutant cells.
It has been determined that PEX11β functions to promote
peroxisome division.
Overall, the paper was
well done. It was well written, and for
the most part the figures were convincing to the reader.
Future Experiments
We now know that the PEX11β
gene is involved in peroxisome division, but it is still not known how the
proteins made from the gene promote division.
How do they interact with other proteins? Li and Gould suggest that perhaps the level of peroxisome division
is dependent on how much PEX11 is concentrated in the membrane. To test this, it would be necessary to test
different concentrations of PEX11 in the membrane. The control would be a wild type cell with normally functioning peroxisomes. A negative control would be a PEX11
deficient mutant with no PEX11 proteins in the membrane. It would be possible to overexpress PEX11 at
different concentration levels in order to test the hypothesis. This could be tested using the same
procedures the writers used in this paper – transfection, microinjection,
immunoflourescence and multiple antibodies.
The authors also suggest that PEX11 proteins are
affected by modification after translation.
In order to determine if this is the case, researchers would simply have
to perform column chromatography. If
PEX11 proteins are modified post-translationally, the protein from the cell
would be a larger molecular weight than if the protein was made in the lab from
the mRNA strand. They could also do
something similar to what we did in lab.
The could make a protein, and if it is nonfunctional, it may be deduced
that perhaps modification takes place in the cell after translation. It is possible that the PEX11 proteins either
modify lipids directly or act to bring other proteins that do modify lipids to
the membrane of the peroxisome. It may
be possible to establish if the other proteins need to bind to the PEX11
proteins in order to work. A nickel
column could determine this.
Cooperative proteins bound together will have a higher weight than just
a PEX11 protein alone or another protein alone.
The authors also suggest that the loss of PEX11 proteins
may physically change the membrane of the peroxisome. A change in the membrane could affect many types of transmembrane
transport systems. This could be
determined by removing PEX11 proteins from a wild type cell and doing FRAP to
see how lipids and proteins are being transported. A control would be a regular wild type cell, where proteins and
lipids move freely. Another would be a PEX11-/-
mutant. This would be a negative
control. Removal of PEX11 proteins
should look like this data. If a
physical change does occur, it would be possible to see a change in the shape
of the peroxisome.
References
Li, X., and S. Gould. 2002. PEX11 promotes peroxisome division
independently of peroxisome metabolism. J. Cell
Biol.156:
643-651
This page is maintained by Nicole Hesson.